Non-typeable Haemophilus influenzae is an important cause of upper and lower respiratory infections in humans. Bacterial adherence to the nasopharyngeal tissue is the first step in colonization. Several non- fimbrial adhesins have been identified in H. influenzae, but the mechanisms of their secretion and regulation have not been elucidated. The broad goal of this project is to determine what role HMW1B plays in the secretion of the HMW1 adhesin. The first specific aim will address the ability of HMW1B to function as a pore for secretion and define the topology and structure of HMW1B. The pore size of HMW1B will be assessed by liposome swelling. Indirectly, HMWB porin activity will be examined via antibiotic susceptibility and the ability of a LamB (a maltoporin) mutant expressing HMW1B to grow on maltodextrins of increasing size. HMW1B topology will be determined by mutational analysis, HMW1B-PhoA protein fusions, epitope tag insertions and protease sensitivity. In the second specific aim, the interactions between HMW1B and HMW1 will be examined. HMW1B interactions with different regions of HMW1 will be explored in a direct ELISA. Exogenous HMW1 will be added to HMW1B-expressing bacterial cells to determine if HMW1 requires HMW1B for cell surface association. HMW1B suppressor mutants that can translocate an HMW1 secretion- deficient mutant will be generated to identify amino acids in HMW1B important for HMW1 translocation.